Osamu Sano

Royal jelly inhibits the production of proinflammatory cytokines by activated macrophages

In this study, we have examined the anti-inflammatory actions of royal jelly (RJ) at a cytokine level. When supernatants of RJ suspensions were added to a culture of mouse peritoneal macrophages stimulated with lipopolysaccharide and IFN-γ, the production of proinflammatory cytokines, such as TNF-α, IL-6, and IL-1, was efficiently inhibited in a dose-dependent manner without having cytotoxic effects on macrophages. This suggests that RJ contains factor(s) responsible for the suppression of proinflammatory cytokine secretion. We named the factor for honeybees RJ-derived anti-inflammatory factor (HBRJ-AIF), and further investigated the molecular aspects of it. Size fractionation study showed that HBRJ-AIF is composed of substances of low (<5 kDa) and high (>30 kDa) molecular weights, with the former being a major component. Chromatographic analysis showed that MRJP3 is one candidate for the HBRJ-AIF with high molecular weights. Thus, our results suggest that RJ has anti-inflammatory actions through inhibiting proinflammatory cytokine production by activated macrophages.

Immunohistochemical and immuno-electron-microscopic detection of interferon-γ-inducing factor (”interleukin-18”) in mouse intestinal epithelial cells

Abstract.The novel cytokine interferon-γ-inducing factor (”interleukin-18”) is produced by macrophage-like cells in mice with endotoxin shock and induces the production of interferon-γ by T cells in vitro. To determine the physiological role for mouse interferon-γ-inducing factor, we studied its tissue distribution in several organs (intestine, spleen, thymus, kidney, and liver) in healthy mice of different ages, including fetal stages. Activity of the cytokine in the organ extracts of adult mice was measured by enzyme-linked immunosorbent assay, and the cellular distribution of interferon-γ-inducing factor in organs from fetal and adult mice was determined by immunohistochemistry. Intestinal extracts of adult mice showed the highest concentrations among the organs studied. Other organ extracts of adult mice showed lower concentrations of the cytokine. Immunohistochemical analysis revealed that interferon-γ-inducing factor was localized in the cytoplasm of intestinal epithelial cells from fetal and adult mice. These results show for the first time that intestinal epithelial cells may be the main producers of interferon-γ-inducing factor under normal physiological conditions and suggest that its constitutive expression in intestinal epithelial cells may have an important role in the induction of mucosal immunity.

Natural Human Interferon-γ Derived from Lipopolysaccharide-stimulated Human Myelomonocytic HBL-38 Cells

A human myelomonocytic cell line, HBL‐38 cells, propagated in vivo, spontaneously produced interferon (IFN)‐γ and IFN‐α. Whereas hemmagglutinating virus of Japan (HVJ) enhanced the production of IFN‐α, bacterial lipopolysaccharide (LPS) markedly enhanced the production of IFN‐γ. LPS could be replaced with lipid A. Furthermore, the enahancement of production of IFN‐γ by LPS was completely abolished by polymixin B. IFN‐γ derived from LPS‐stimulated HBL‐38 cells was purified to homogeneity and characterized. The apparent molecular weight, subspecies composition, amino acid sequence and glycosylated sites were in agreement with those of the product of normal human peripheral blood lymphocytes (PBL). These results indicate that the myelomonocytic HBL‐38 cells, not a T‐cell line, can also produce IFN‐γ identical to the product of normal human PBL.

Inhibitory Effects of 2-Amino-3H-phenoxazin-3-one on the Melanogenesis of Murine B16 Melanoma Cell Line

Hyperpigmentations are a serious concern addressed by both the medical community and the cosmetic industry through the development of agents that block melanin biosynthesis. In this study, we found that 2-amino-3H-phenoxazin-3-one (APO), isolated from extracts of the edible mushroom Agaricus bisporus Imbach, exhibited potent inhibitory effects on melanogenesis in B16 cells, a murine melanoma cell line. APO inhibited melanin biosynthesis at 1,000 times lower concentrations (IC50=1.31±0.08 μM) than kojic acid (IC50=1.31±0.13 mM), without causing cellular toxicity. APO did not directly inhibit the enzyme activity of tyrosinase, the rate-limiting melanogenic enzyme. Further study showed that APO inhibited the protein expression of tyrosinase and microphthalmia-associated transcription factor (MITF), a melanogenic transcription factor that regulates the expression of tyrosinase. These results suggest that APO is a promising depigmenting agent with both therapeutic and cosmetic value in preventing melanogenesis.

Anti-inflammatory effects of adenosine N1-oxide.

BackgroundAdenosine is a potent endogenous anti-inflammatory and immunoregulatory molecule. Despite its promise, adenosine’s extremely short half-life in blood limits its clinical application. Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly. ANO is an oxidized product of adenosine at the N1 position of the adenine base moiety. We found that it is refractory to adenosine deaminase-mediated conversion to inosine. We further examined the anti-inflammatory activities of ANO in vitro and in vivo.MethodsThe effect of ANO on pro-inflammatory cytokine secretion was examined in mouse peritoneal macrophages and the human monocytic cell line THP-1, and compared with that of adenosine, synthetic adenosine receptor (AR)-selective agonists and dipotassium glycyrrhizate (GK2). The anti-inflammatory activity of ANO in vivo was examined in an LPS-induced endotoxin shock model in mice.ResultsANO inhibited secretion of inflammatory mediators at much lower concentrations than adenosine and GK2 when used with peritoneal macrophages and THP-1 cells that were stimulated by LPS plus IFN-γ. The potent anti-inflammatory activity of ANO could not be solely accounted for by its refractoriness to adenosine deaminase. ANO was superior to the synthetic A1 AR-selective agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), A2A AR-selective agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5’-N-ethylcarboxamideadenosine hydrochloride (CGS21680), and A3 AR-selective agonist, N6-(3-iodobenzyl)adenosine-5’-N-methyluronamide (IB-MECA), in suppressing the secretion of a broad spectrum of pro-inflammatory cytokines by peritoneal macrophages. The capacities of ANO to inhibit pro-inflammatory cytokine production by THP-1 cells were comparable with those of CCPA and IB-MECA. Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock. A significant increase in survival rate was also observed by oral administration of ANO. Mechanistic analysis suggested that the up-regulation of the anti-inflammatory transcription factor c-Fos was, at least in part, involved in the ANO-induced suppression of pro-inflammatory cytokine secretion.ConclusionsOur data suggest that ANO, a naturally occurring molecule that is structurally close to adenosine but is functionally more potent, presents potential strategies for the treatment of inflammatory disorders.